This invention relates to a method of stimulating ciliary beat frequency to promote mucociliary or cough clearance of retained mucus secretions from the lungs, sinuses, or ears of a patient by administering certain uridine, adenosine, or cytidine triphosphates.
Mucociliary clearance is an important defense mechanism of the human airway and middle/inner ear tract. Coordinated beats of cilia in the nose, trachea, bronchi, and middle ear propel the mucous layer toward the pharynx, carrying along with it microorganisms and other particles captured in the mucus. Normal function of this system depends on the frequency and coordination of ciliary beating and the properties of mucus. There are three components of the mucociliary clearance system: (1) the mucin layer, which is formed by secretion of mucins by goblet cells, (2) cilia, which transport the overlying mucin layer by synchronous beating, and (3) the periciliary liquid layer, which surrounds the cilia and is less viscous than the mucin layer, allowing free movement of the cilia. The electrolyte and water concentration of the periciliary layer is regulated by the luminal epithelial cells. (R. Boucher, et al., Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology, p. 525-32 entitled xe2x80x9cMechanisms and Therapeutic Actions of Uridine Triphosphates in the Lungxe2x80x9d (L. Belardinelli, et al. ed., Alumwer Academic Publishers, Boston 1995)).
Primary ciliary dyskinesia (PCD) is a congenital disease characterized by ultrastructural defects and motility disturbances of cilia, resulting in either absent or abnormal ciliary movement. The most common clinical manifestations of PCD are chronic respiratory disease (e.g., sinusitis, rhinitis, and bronchiectasis) and otitis media. Because PCD patients have either absent or severely impaired mucociliary clearance (MCC), the only available mechanism to clear or move secretions is cough. Cough clearance may be measured in a manner similar to that previously described for MCC. PCD also impairs the propulsion of spermatozoa, resulting in male infertility. (D. Schidlow, Ann Alergy 73(b), 457-68 (1995)). PCD also results in the impairment of cell motility of certain immune system cells, including neutrophils and macrophages. (N. Valerius, Eur j Clin Invest 13, 489-94 (1983)). PCD may be responsible for a form of hydrocephalus caused by ciliary malfunction. (M. Greenstone, Arch Dis Child 59, 481-82 (1984)). The incidence of PCD has been calculated to be one in 16,000 live births, and an estimated 50% of affected individuals also have situs inversus (dextrocardia). The triad of bronchiectasis, sinusitis, and situs inversus (dextrocardia) is referred to as Kartagener""s syndrome. (M. Sleigh, Lancet ii, 476 (1981)). It has been hypothesized that Kartagener""s syndrome is caused by a lack of embryonic ciliary movement, resulting in the random rotation, of the archenteron such that in half the cases there is situs inversus (dextrocardia) and in the other half there is normal cardia situs. (B. Afzelius Science 193, 317-19 (1976)). The clinical course of PCD is characterized primarily by sinus and ear infections early in life with a progressive change to lung/lower airways diseases in adulthood. Chronic airways infections can lead to chronic obstructive changes in the pulmonary tissue, progressive loss of pulmonary function, and eventually death.
A secondary and more common form of ciliary dyskinesia is the acquired form of the disease. Chronic inflammation caused by severe viral or bacterial respiratory infections, chronic smoking, severe air pollution, chemical or thermal burns to the airways, intubation and mechanical ventilation, and near drowning can result in changes in ciliary structure including disruption of the cellular membrane, loss or incorporation of microtubules, and formation of compound cilia, all of which can result in abnormal or absent ciliary function. (J. Ballenger Ann Otol Rhinol Laryngol 97 (3 Pt. 1), 253-58 (1988); M. Pedersen Lung 168 Suppl., 368-76 (1990)). Respiratory infections which often lead to secondary ciliary dyskinesia include influenza, adult respiratory distress syndrome, and ventilator-associated pneumonia (VAP) in intensive care unit (ICU) patients. In some cases acquired ciliary dyskinesia may be reversed with appropriate and timely intervention; however, permanent damage and/or sustained exposure to the above factors may render the ciliary damage irreversible. The clinical manifestations and course would likely appear similar to PCD with respect to chronic lung infections, progressive loss of pulmonary function, and obstructive pulmonary disease.
The typical mammalian respiratory epithelial cell contains about 200 cilia. Each cilium has nine peripheral microtubular doublets and two central tubules. Each peripheral doublet contains an A subunit and a B subunit, and each A subunit has a set of curved arms attached to it called the inner and outer dynein arms. These dynein arms contain ATPase-an enzyme which breaks down adenosine triphosphate (ATP), providing the energy for ciliary movement. Because the most common ultrastructural abnormality associated with primary ciliary dyskinesia is the total absence of dynein arms (B. Afzelius, et al., J Cell Biol 66, 225-32 (1975)), researchers began investigating whether extracellular application of ATP and ATPase could activate immotile cilia in vitro. (J. Forrest, et al., Am Rev Resp Dis 120, 511-15 (1979)). Although the results appeared positive, the findings have not been consistently reproduced by others. It was later discovered that extracellular application of Ca2+ and cAMP could increase the beat frequency of respiratory tract cilia. (A. Lansley, et al., Am J. Physiol 263, L232-42 (1992)). It has not been definitively established that any therapy will stimulate cilia beat in cases where complete ciliary immotility has been demonstrated. In such cases, it might be of therapeutic benefit to increase hydration of the viscous mucous secretions.
Applicant has discovered that extracellular triphosphate nucleotides, especially uridine 5xe2x80x2-triphosphate (UTP) modulates all three components of the mucociliary transport system. UTP stimulates ciliary beat frequency (R. Boucher, et al., supra); UTP stimulates mucin secretion by goblet cells (M. Lethem, et al., Am J Respir Cell Mol Biol 9, 315-22 (1993)); and UTP stimulates Cl- secretion in airway epithelial cells, which increases hydration of the periciliary liquid layer (M. Knowles, et al., N Eng j. Med 325, 533-38 (1991)). Applicant has also demonstrated that UTP is safe and improves cough clearance in PCD patients. (P. Noone, et al., abstract submitted to the 1996 International Conference of The American Thoracic Society).
In summary, a variety of clinical manifestations of ciliary dyskinesia, such as absent or impaired mucociliary clearance in the respiratory and middle/inner ear tract, impaired propulsion of spermatozoa, and impaired motility of neutrophils and macrophages can be improved or alleviated by administering UTP and its related compounds, as well as other nucleoside phosphates such as: adenosine 5xe2x80x2-triphosphate (ATP); cytidine 5xe2x80x2-triphosphate (CTP); 1,N6-ethenoadenosine triphosphate; adenosine 1-oxide triphosphate; 3,N4-ethenocytidine triphosphate; P1,P4-di(adenosine-5xe2x80x2) tetraphosphate (A2P4); or P1, P4-di(uridine-5xe2x80x2) tetraphosphate (U2P4) to the affected part of the body.
A method of treating ciliary dyskinesia in a subject in need of such treatment is disclosed. The method comprises administering to the patient a compound of Formula I, or a pharmaceutically acceptable salt thereof, in an amount effective to stimulate ciliary beat frequency, where possible, in the luminal epithelial cells of the lung or middle/inner ear, eyes, genito-urinary tract; spermatozoa cells; or certain cells of the immune system, including neutrophils and macrophages: 
wherein:
X1, X2, and X3 are each independently either Oxe2x88x92 or Sxe2x88x92. Preferably, X2 and X3 are Oxe2x88x92.
R1 is O, imido, methylene, or dihalomethylene (e.g., dichloromethylene, diflouromethylene). Preferably, R1 is oxygen or difluoromethylene.
R2 is H or Br. Preferably, R2 is H. Particularly preferred compounds of Formula I are uridine 5xe2x80x2-triphosphate (UTP) and uridine 5xe2x80x2-O-(3-thiotriphosphate) (UTPxcex3S).
Formula I is the preferred embodiment of the compound, however, the method of the present invention can also include administering a compound of Formula II (adenosine 5xe2x80x2 triphosphate [ATPA ] or 1,N6-ethenoadenosine triphosphate or adenosine 1-oxide triphosphate), or Formula III (cytidine 5xe2x80x2 triphosphate [CTP] or 3,N4-ethenocytidine triphosphate), or Formula IV (P1,P4-di(adenosine-5xe2x80x2) tetraphosphate (A2P4) or p1,p4 di(uridine-5xe2x80x2) tetraphosphate (U2P4). 
wherein:
R1, X1, X2, and X3 are defined as in Formula I.
R3 and R4 are H while R2 is nothing and there is a double bond between N-1 and C-6 (adenine), or
R3 and R4 are H while R2 is O and there is a double bond between N-1 and C-6 (adenine 1-oxide), or
R3, R4 and R2 taken together are xe2x80x94CHxe2x95x90CHxe2x80x94, forming a ring from N-6 to N-1 with a double bond between N-6 and C-6 (1,N6-ethenoadenine). 
xe2x80x83wherein:
R1, X1, X2, and X3 are defined as in Formula I.
R5 and R6 are H while R7 is nothing and there is a double bond between N-3 and C-6 (cytosine), or,
R5, R6 and R7 taken together are xe2x80x94CHxe2x95x90CHxe2x80x94, forming a ring from N-3 to N-4 with a double bond between N-4 and C-4 (3,N4-ethenocytosine). 
wherein:
B is adenine or uracil.
A second aspect of the present invention is a pharmaceutical formulation containing the compound of Formula I, I, III, or IV in an amount effective to stimulate ciliary beat frequency in: the epithelial cells of the lungs or middle and inner ears; the mucous clearance defense system of the eyes or genito-urinary tract; spermatozoa cells; the ovaries or fallopian tubes; or certain cells of the immune system, including at neutrophils and macrophages, in a pharmaceutically acceptable carrier.
A third aspect of the present invention is the use of the active compounds disclosed herein for the manufacture of a medicament for the therapeutic stimulation of ciliary beat frequency in: the epithelial cells of the lungs or middle and inner ears; the mucous clearance defense system of the eyes or genito-urinary tract; spermatozoa cells; the ovaries or fallopian tubes; or certain cells of the immune system, including neutrophils and macrophages, of a patient in need of such treatment.
The method of the present invention may be used to stimulate ciliary beat frequency in a subject in need of such treatment for any reason, including (but not limited to) increasing the mucociliary clearance of retained secretions in the lungs, sinuses, or middle and inner ears. The method of the present invention may also be used to treat primary ciliary dyskinesia, secondary ciliary dyskinesia, Kartagener""s syndrome, otitis media, cystic fibrosis, diseases involving the dysfunction of the ocular or genito-urinary mucociliary clearance defense system caused by impairment of ciliary movement, diseases of the immune system caused by impairment of ciliary movement of neutrophils and macrophages, hydrocephalus caused by impairment of ciliary movement, male infertility caused by impairment of the ciliary propulsion of the spermatozoa, female infertility caused by impairment of ciliary movement on the luminal epithelial cells of the ovaries or fallopian tubes, or any other disease caused by an impairment of ciliary movement. The present invention increases mucociliary clearance in three ways: (1) by increasing the ciliary beat frequency of cilia on the surface of luminal epithelial cells, (2) by increasing the secretions of mucins by goblet cells, and (3) by increasing the secretion of water into the periciliary liquid layer by luminal epithelial cells. The mucins secreted by goblet cells form a layer on top of the cilia and captures foreign particles, including viruses and bacteria; the mucin layer is transported by the wave-like action of cilia, and the movement of cilia is facilitated by the composition and hydration of the periciliary liquid layer surrounding the cilia. Although the primary aspect of the present invention is to increase ciliary beat frequency in patients afflicted with ciliary dyskinesia, in patients whose cilia are permanently incapable of any movement regardless of treatment, the active compounds of the present invention can still facilitate the clearance of retained mucous secretions by increasing the secretion of water into the periciliary liquid layer and by increasing the secretion of mucins by goblet cells.
Additionally, because of the well-demonstrated ability of the active compounds of the present invention to enhance lung mucociliary clearance in normal subjects, the active compounds of the present invention can accelerate the clearance of any type of inhaled foreign materials from the airways. This would prove beneficial in a number of situations-biological warfare, e.g. the chemical warfare agent ricin; smoke inhalation; industrial exposure to inhaled toxins (resulting in e.g., silicosis, anthracosis, and the gamut of so-called pneumoconioses); and allergic reaction to inhaled particles such as pollen.
Furthermore, the ability of the active compounds of the present invention to increase lung clearance would also prove beneficial in the diagnosis of lung disease-specifically, to improve the quality of radioisotopic scans of the lungs by removing the secretions that might otherwise obscure the visualization of ventilated portions of the lung. In radioisotopic lung scanning, the mismatch of ventilated versus perfused lung is used to identify areas of pulmonary infarction. As a result of improved aeration of the lungs after administering the active compounds of the present invention, the ventilated portions of the scan would be more distinct, and the diagnostician would be in a better position to clearly identify true mismatches.
The present invention is concerned primarily with the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes.
Compounds illustrative of the compounds of Formula I above include: (a) uridine 5xe2x80x2-triphosphate (UTP); (b) uridine 5xe2x80x2-O-(3-thiotriphosphate) (UTPxcex3S); and (c) 5bromo-uridine 5xe2x80x2-triphosphate (5-BrUTP). These compounds are known or may be made in accordance with known procedures, or variations thereof which will be apparent to those skilled in the art. See generally N. Cusack and S. Hourani, Annals N.Y. Acad. Sci. 603, 172-81 (entitled xe2x80x9cBiological Actions of Extracellular ATPxe2x80x9d). For example, UT? may be made in the manner described in Kenner, et al., J. Chem. Soc. 1954, 2288; or Hall and Khorana, J. Am. Chem. Soc. 76, 5056 (1954). See Merck Index, Monograph No. 9795 (11th Ed. 1989). UTPxcex3S may be made in the manner described in R. S. Goody and F. Eckstein, J. Am. Chem. Soc. 93, 6252 (1971).
For simplicity, Formulae I-IV herein illustrate the active compounds in the naturally occuring D-configuration, but the present invention also encompasses compounds in the L-configuration, and mixtures of compounds in the D- and L-configurations, unless otherwise specified. The naturally occuring D-configuration is preferred.
Compounds illustrative of the compounds of Formula II above include (a) adenosine 5xe2x80x2-triphosphate (ATP) and (b) 1,N6-ethenoadenosine triphosphate. Compounds illustrative of the compounds of Formula m above include (a) cytidine 5xe2x80x2-triphosphate and (b) 3,N4-ethenocytidine triphosphate. These compounds can be made in accordance with known procedures, or variations thereof which will be apparent to those skilled in the art. For example, phosphorylation of nudeosides by standard methods such as D. Hoard and D. Ott, J. Am. Chem. Soc. 87, 1785-1788 (1965); M. Yoshikawa, et al., Tetrahedron Lett. 5065-68 (1967) and idem., Bull. Chem. Soc. (Jpn) 42, 3505-08 (1969); J. Moffatt and H. Khorana, J. Am. Chem. Soc. 83, 649-59 (1961); and B. Fischer, et al., J. Med. Chem. 36, 3937-46 (1993) and references therein. Etheno derivatives of cytidine and adenosine are prepared by known methods such as: N. Kotchetkov, et al., Tetrahedron Lett. 1993 (1971); J. Barrio, et al., Biochem. Biophys. Res. Commun. 46, 597 (1972); J. Secrist, et al., Biochemistry 11, 3499 (1972); J. Bierndt, et al., Nucleic Acids Res. 5, 789 (1978); K. Koyasuga-Mikado, et al., Chem. Pharm. Bull. (Tokyo) 28, 932 (1980). Derivatives with alpha, beta and gamma thiophosphorus groups can be derived by the following or by adapting methods of: J. Ludwig and F. Eckstein, J. Org. Chem. 54, 631-35 11989); F. Eckstein and R. Goody, Biochemistry 15, 1685 (1976); R. Goody and F. Eckstein, J. Am. Chem. Soc. 93,6252 (1971).
Compounds of Formulas I, II, or m where R1 is CCl2 and CF2 can be prepared by methods similar to that described in G. Blackburn, et al., J. Chem. Soc. Perkin Trans. I, 1119-25 (1984). Compounds of Formula I, II, III where R1 is CH2 can be prepared by methods similar to that described in T. Myers, et al., J. Am. Chem. Soc. 85, 3292-95 (1963).
Compounds illustrative of the compounds of Formula IV include (P1,P4-di(adenosine-5xe2x80x2) tetraphosphate (A2P4) or p1,p4-di(uridine-5xe2x80x2) tetraphosphate (U2P4). These compounds can be made in accordance with known procedures, or variations thereof which will be described by: P. Zamecnik, et al., Proc. Nati. Acad. Sci. USA 89, 838-42 (1981); and K. Ng and L. E. Orgel, Nucleic Acids Res. 15 (8), 3572-80 (1987).
In addition, UTP, ATP, CTP, A2P4, 3,N4-ethenocytidine triphosphate, 1,N6-ethenoadenine triphosphate, adenosine 1-oxide triphosphate, ATPxcex3S, ATPxcex2S, ATPxcex1S, AMPPCH2P, AMPPNHP, N4-ethenocytidine and 1,N6-ethenoadenosine are commercially available, for example, from Sigma Chemical Company, PO Box 14508, St. Louis, Mo. 63178.
The active compounds of Formulae I-IV may be administered by themselves or in the form of their pharmaceutically acceptable salts, e.g., an alkali metal salt such as sodium or potassium, an alkaline earth metal salts such as manganese, magnesium and calcium or an ammonium and tetraalkyl ammonium salts, NX4+ (wherein X is C1-4 alkyl group). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.
The active compounds disclosed herein may be administered to the lungs, sinuses, ears, eyes, spermatozoa, ovaries or fallopian tubes, or genito-urinary tract by a variety of suitable means, but are preferably administered by administering a nebulized form of the active compound into their respiratory tract, such that the active compound enters the lungs and reaches the area of the body afflicted with impaired ciliary movement either directly or via systemic absorption and circulation. The active compound can be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalants, metered dose inhalants, or liquid/liquid suspensions. In dry powder delivery, the UTP may be formulated alone or in combination with diluent or carrier, such as sugars (e.g., lactose, sucrose, trehalose, mannitol) where the compounds may be intimately incorporated in the matrix through glassification or simply admixed with .the carrier, or other acceptable excipients for lung or airway delivery. The dry powder may be obtained by methods known in the art, such as spray-drying, milling, freeze-drying, super-critical fluid manufacturing or via controlled crystallization or precipitation
The dosage of active compound to stimulate ciliary beat frequency will vary depending on the condition being treated and the state of the subject, but generally an effective amount is the amount sufficient to achieve concentrations of active compound on the lungs, sinuses, ears, eyes, or genito-urinary surfaces of the subject of from about 10xe2x88x927 to about 10xe2x88x921 Moles/liter, and more preferably from about 10xe2x88x926 to about 10xe2x88x921Moles/liter.
Depending upon the solubility of the particular formulation of active compound administered, the daily dose to promote fluid drainage may be divided among one or several unit dose administrations. Preferably, the daily dose is no more than four times per day.
Another means of administering the active compound to the lungs, sinuses, ears, eyes, or genito-urinary tract of the patient to promote fluid/secretion drainage may include any oral form of the active compound, administered to the patient either by means of a liquid suspension of the active compound which is poured into the mouth of the patient, or by means of a pill form swallowed by the patient.
Another means of administering an effective amount of the active compound to the lungs, sinuses, eyes, or middle and inner ears would involve administering a liquid/liquid suspension (either a nasal spray of respirable particles which the subject inhales, or nasal drops of a liquid formulation, or eye drops of a liquid formulation) comprised of the active compound. Liquid pharmaceutical compositions of the active compound for producing a nasal spray or nasal or eye drops may be prepared by combining the active compound with a suitable vehicle, such as sterile pyrogen free water or sterile saline by techniques known to those skilled in the art.
Another means of administering the active compound to the middle ear or eye would include any topical form of the active compound, administered as a cream or gel to the outer ear or eye, which would subsequently permeate through the tympanic membrane or cornea into the middle ear or lens of the patient.
Another means of administering the active compound to the middle ear, genito-urinary tract, or eye would involve an injected form of the active compound, injected from the outer ear directly through the tympanic membrane into the middle ear, injected into the genito-urinary tract, or injected into the eye. This could involve a patch containing UTP which would be applied directly to the tympanic membrane.
Another means of administering the active compound to the lungs, sinuses, middle and inner ears, eyes, or genito-urinary tract would involve a suppository form of the active compound, such that a therapeutically effective amount of the compound reaches the lungs, sinuses, middle ear, eye, genito-urinary tract, or male or female reproductive systems via systemic absorption.
Another means of administering the active compound would involve intra-operative instillation of a gel, cream, or liquid suspension form of the active compound such that a therapeutically effective amount reaches the lungs, sinuses, middle and inner ears, eyes, or genito-urinary tract.
An additional means of administering the active compound would involve ex vivo administration of the active compound to the spermatozoa by means of a topical, injection, or immersion form of the compound, such that a therapeutically effective amound of said compound contacts the spermatozoa having impaired impaired ciliary movement.
An additional means of administering the active compound would involve administration of the active compound via a transdermal patch, in which the active compound would be delivered to the affected are via local absorption or systemic absorption and circulation.
UTP and compounds for Formulae I-IV also have therapeutic benefit when used in combination with other agents used to treat ciliary dyskinesia, such as, but not limited to: antibiotics; vaccines; decongestants, mucolytic agents; nonsteroidal antiinflammatory agents; steroids; antiviral agents; and bronchodilators. UTP may also be used in combination with other treatments under development, such as gene therapy. UTP may also be used in combination with the recently discovered therapeutic protein defensin.